Este trabajo fue apoyado por el Instituto Canadiense de Investigación en Salud. CSC fue financiado por una beca postdoctoral de las Ciencias Naturales e Ingeniería de Investigación de Canadá. FD es un Tier II Cátedra de Investigación de Canadá.

<html> Proceso de reconstitución global El proceso de reconstitución se inicia mediante la mezcla de la proteína de membrana andamio (MSP) con el complejo purificado MalFGK 2 en presencia de solubilizadas en detergente fosfolípidos. El paso es seguido por la eliminación lenta del detergente por un material de poliestireno adsorbente llamado Bio-Beads o Amberlite (Figura 1). El proceso de auto-ensamblaje ocurre muy probablemente debido a las interacciones apolares entre los fosfolípidos hidrófobos, el complejo MalFGK 2 y la superficie de la proteína anfipática MSP. El producto final es una partícula discoide formado por dos moléculas de envolver alrededor de la MSP 2 MalFGK complejo. Las partículas se separan de los aductos y los agregados por ultra-centrifugación y cromatografía analítica de exclusión por tamaño. Las partículas se caracterizan por electroforesis en gel nativo y espectroscopia de dispersión de luz dinámica. título &quot;&gt; 1. preparación de la membrana andamio de proteínas, MSP <ol><li> El Su-etiquetados MSP (versión MSP1D1 1) se produce a partir pMSP1D1 plásmido en E. coli BL21 (DE3) indujo a una DO de 600 ~ 0,5 con 0,5 mM de isopropil β-D-1-tiogalactopiranósido durante 3 horas a 37 ° C. </li><li> Las células se recogieron por centrifugación a 5.000 xg durante 10 min a 4 ° C y se resuspendieron en tampón que contenía TSG10 100 mM fluoruro de fenilmetanosulfonilo. </li><li> Las células se lisaron con una prensa francesa (3x) a 8.000 psi y el material insoluble se eliminó por centrifugación a 5.000 xg durante 10 min a 4 ° C. La fracción soluble que contiene el MSP se aísla por ultra-centrifugación a 125.000 xg durante 45 min. </li><li> El MSP se purifica mediante cromatografía de níquel quelante de uso de ~ 1,5 ml Ni Sepharose HP en TSG10 buffer. Los contaminantes se eliminan por lavado con TSG10 tampón que contiene 50 mM de imidazol. El MSP se eluye con tampón que contiene TSG10 600 mMimidazol. La proteína purificada se dializó en tampón TSG10 y almacenado en -70 º C a una concentración de proteína de ~ 10-15 mg / ml. </li></ol> 2. Preparación de la MalFGK 2 Complejo <ol><li> El MalFGK 2 compleja, su etiquetado en el extremo C-terminal de Malk, se expresa a partir del plásmido pBAD22-FGK en E. coli BL21 (DE3) y se indujo a una DO de 600 ~ 0,5 con 0,2% de L-arabinosa durante 3 horas a 37 ° C. </li><li> Después de la lisis celular y centrifugación como en el paso 1,3, el sedimento de membrana se resuspende en tampón TSG20 a una concentración final de 5 mg / ml. El material se solubiliza con w / v 1% de n-dodecil-β-maltopiranósido (DDM) durante 3 horas a 4 ° C con agitación suave. </li><li> El material insoluble se elimina por ultra-centrifugación como en el paso 1,3 y el sobrenadante que contiene el solubilizado 2 MalFGK complejo se recogió y se purificó como en el paso 1,4, pero sin diálisis. </li><li> Further purificación se realizó mediante cromatografía de filtración en gel en tampón TSGD en un Superdex 200 HR 10/300 columna a un caudal de 0,5 ml / min. </li></ol> 3. Preparación de fosfolípidos <ol><li> Una E. lípidos totales coli extracto disuelto en cloroformo se separó en alícuotas de 1.000 nmol en tubos de microcentrífuga tornillos de cabeza. El disolvente se evaporó bajo una corriente suave de nitrógeno y se secó adicionalmente durante una noche en un desecador a vacío. </li><li> La película de lípido se disuelve en tampón TS a una concentración de 5 nM final y vortex vigorosamente, y se sonicó. Los lípidos disueltos aparecerá ligeramente opaco debido a su insolubilidad en general en tampón TS acuoso, pero debe permanecer en suspensión. DDM se añade a una concentración final de 0,5% (~ 10 mM), en la que la solución se vuelva clara. </li><li> La mezcla de lípidos se coloca en un baño de agua y el pulso se sonicó durante 5 veces para ~ 5 seg. La mezcla de lípidos se almacenan a 4 ° C durante un máximo de 1 week. </li></ol> 4. Preparación de Bio-Beads <ol><li> Aproximadamente el 10-15 ml (volumen seco) Bio-Beads se coloca en un tubo de 50 ml. </li><li> Las perlas se lavaron sucesivamente con 50 ml de metanol al 100%, 95% de etanol, milliQ H 2 O, y finalmente tampón TS (dos veces cada uno). </li><li> Las perlas lavadas se almacenan a 4 ° C en tampón de ~ 10 ml de TS. </li></ol> 5. Nanodisc Reconstitución <ol><li> El MSP se diluye en tampón TSGD a la concentración final de ~ 7 mg / ml (~ 0,3 mM). </li><li> ~ 2 nmol de 2 purificado MalFGK complejo se mezclan a una proteína: MSP: lípido de 1:3:60 o 1:3:400 en tampón TSGD. La concentración final es de 6 micras MalFGK 2, 18 mM y lípidos MSP 360 micras (1:3:60) o 2,4 mM (lípidos 1:3:400). El volumen final es de 300 l. La concentración final de DDM es 0,08% (~ 1,6 mM). La concentración final de glicerol depende de la cantidad de lípido añadido, pero sigue siendo alrededor de 5-10% v / v </li&gt; <li> ~ 50 l de Bio-Bead suspensión se añadió al tubo y se incuba la mezcla durante toda la noche en un agitador a 4 º C. </li><li> Las perlas se sedimentan por gravedad y la solución se pipeteó a través de una punta estrecha para evitar en la medida Bio-Beads como sea posible. </li><li> Grandes precipitados se separan por ultra-centrifugación a 100.000 xg durante 20 min. Los discos se purificó por cromatografía de filtración en gel como en el paso 2,4 en TSG10 tampón. Las fracciones que contenían los discos se reúnen y una alícuota se vuelve a inyectar en la columna de filtración en gel mismo para probar la estabilidad de la preparación. </li><li> Los discos purificadas pueden almacenarse a -70 ° C durante un período prolongado de tiempo. Después de la descongelación en hielo, los discos debe ser sometido a ultra-centrifugación como en el paso 5,5 para eliminar el potencial de precipitados. Una alícuota se analizó por cromatografía de filtración en gel para garantizar que la agregación o precipitación significativa no se produjo durante la descongelaciónproceso. </li></ol> 6. Electroforesis en gel nativo <ol><li> 1 M de nanodiscos (~ 0,2 mg / ml) se analizan por PAGE nativa 6, 7 (Tris-HCl pH 8,8, 4-12%) para evaluar la calidad de la reconstitución (Figura 3A). </li><li> La unión de macho (1 M) a la 2-complejo MalFGK reconstituyó en nanodiscos también es evaluado por nativo-PAGE (Figura 3A). </li><li> Después de la electroforesis, el gel se tiñe con azul de Coomassie durante 10 min, y se decoloró durante ~ 1 hr. </li></ol> 7. Dispersión de luz dinámica (DLS) <ol><li> Nanodiscos se purificó por cromatografía de filtración en gel como en el paso 2,4 en TSG10 tampón utilizando un Superdex 200 HR 10/300 columna a un caudal de 0,1 ml / min. Las fracciones que contenían nanodiscos se reúnen y se concentran a ~ 10 mg / ml con un filtro Amicon centrífuga. </li><li> La muestra se filtra dos veces (0,22 micras filtro) antes del análisis por DLS utilizandoun instrumento DynaPro NANOSTAR (Wyatt Technology) en un volumen de 1 l cubeta de cuarzo interno. Los datos se ajustaron usando el software de dinámica (Wyatt Technology) para estimar el diámetro y el peso molecular de las partículas. </li></ol> 8. ATPasa Medidas <ol><li> La actividad de ATPasa de MalFGK 2-reconstituidas nanodiscos se determina mediante un ensayo colorimétrico 8. </li><li> 1 M de discos purificadas y 1 mM de ATP se mezclan con cantidades crecientes de MalE y se incubaron a 37 ° C durante 20 min. La liberación de fosfato inorgánico se midió a 660 nm. </li><li> La cantidad de fosfato liberado se comparó con una curva estándar generada con una solución estándar de fósforo. </li></ol> 9. Los resultados representativos Los nanodiscos se purificó por cromatografía de filtración en gel (Figura 2A, izquierda). El cromatograma muestra que la mayoría de la reconstituida dISCS (negro traza) se eluyen como un solo pico, mientras que los discos hechos con exceso de lípidos (curva roja) eluyen en el volumen vacío y como una serie de picos anchos. La calidad de los nanodiscos se analiza adicionalmente por electroforesis en gel nativo y espectroscopia de dispersión de luz dinámica (DLS). Reconstituidos correctamente los discos migran como una banda fuerte en el gel mientras que los reconstituida en presencia de un exceso de lípidos migrar como frotis (Figura 2A, derecha). El análisis por DLS muestra que la población disco es homogéneo con un diámetro medio de 11,4 nm (Figura 2B). Los discos reconstituidas tienen un peso molecular aparente de 215 kDa basado en la aproximación DLS. Las muestras reconstituidas en la presencia de exceso de lípidos de visualización ampliamente distribuido radios alrededor de 100 nm, que es típico de las muestras no homogéneas. La calidad de la MalFGK 2 complejo se evaluó por electroforesis en gel nativo y su actividad por ATPasalas mediciones (Figura 3). El macho proteína de unión a maltosa se ​​une con una afinidad alta a la MalFGK 2 transportador 9, 10. Utilizando electroforesis no desnaturalizante en gel, es posible detectar un complejo entre el macho y MalFGK 2 (Figura 3A). La estimulación de la actividad de la ATPasa MÄLK 2 por MalE se muestra en la Figura 3B. <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo típico para el protocolo de reconstitución. <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Figura 2"/> Figura 2. Control de calidad de la preparación nanodisc. A. Análisis de filtración en gel (Superdex 200 HR 10 / columna de 300) de los nanodiscos reconstituido en relación baja en lípidos (01/03/60; trazo negro) o la relación de lípido alta (1/3/400; trazo rojo). Electroforesis en gel nativo de la preparación del disco mismo. Molecular marcadores de peso en kDa se indican. B. La luz dinámica de análisis de dispersión de la preparación de un mismo disco. <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Figura 3"/> Figura 3. Análisis de la MalFGK 2-nanodisc partículas. A. Análisis Gel cambio de MalE incubaron con cantidades crecientes de MalFGK 2 nanodisc partículas. B. actividad ATPasa de la MalFGK 2-nanodisc partículas como una función de la concentración masculina.

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No hay conflictos de interés declarado.

Se describe un procedimiento sencillo para la reconstitución del transportador de maltosa en nanodiscos. El transportador es ATPasa activa y la interacción con el macho pareja de unión soluble puede ser recreada (Figura 3). La reconstitución exitosa del transportador en nanodiscos abrir el camino para un análisis adicional biofísica y bioquímica. De particular interés será el análisis sistemático de la ATPasa y la actividad Malk maltosa transporte en el detergente, liposomas y nanodiscos. Tra...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Amicon Ultra-4 50K filtro centrífugo </td><td> Millipore </td><td> UFC805008 </td><td> Seguir el protocolo del fabricante para el uso apropiado </td></tr><tr><td> Bio-Beads SM-2 Adsorbente </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli lípidos totales </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Disuelto en cloroformo, según sea apropiado para manejar un disolvente orgánico </td></tr><tr><td> Ni Sepharose HP resina </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Fósforo solución estándar </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE Healthcare </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla I. Los reactivos específicos. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Nombre </td><td> Composición </td><td> Comentarios </td></tr><tr><td> DDM acciones </td><td> 10% w / v DDM </td><td> Volver a suspender en agua MilliQ y se almacena a -20 ° C </td></tr><tr><td> MalFGK 2 stock </td><td> 1-2 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v de glicerol 0,03% w / v DDM </td><td> Almacenar a -70 ° C después de la purificación </td></tr><tr><td> MSP acciones </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v de glicerol </td><td> Almacenar a -70 ° C después de la purificación en &lt;alícuotas de 1 ml y evitar excesivos ciclos descongelación / congelación </td></tr><tr><td> Fosfolípidos acciones </td><td> 5 nM E. coli total de lípidos 0,5% w / v (10 mM) DDM 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Almacenar a 4 ° C durante 1 semana </td></tr><tr><td> TS búfer </td><td> 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Almacenar a 4 ° C </td></tr><tr><td> TSG10 búfer </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v de glicerol </td><td> Almacenar a 4 ° C </td></tr><tr><td> TSG20 búfer </td><td> 50 mM Tris-HCl, pH 8 100 mM NaCl 20% v / v de glicerol </td><td> Almacenar a 4 ° C </td></tr><tr><td> TSGD búfer </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v de glicerol 0,03% w / v DDM </td><td> Almacenar a 4 ° C y se añade justo antes de utilizar DDM </td></tr><tr><td colspan="3"> Tabla II. Recetas de soluciones. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

El nanodisc es una partícula discoidal (~ 10-12 nm grande) que las proteínas de membrana trampa en un pequeño parche de doble capa de fosfolípidos. El nanodisc es una opción particularmente atractiva para el estudio de proteínas de membrana, especialmente en el contexto de las interacciones ligando-receptor. El método por primera vez por Sligar y colegas se basa en las propiedades anfipáticas de una ingeniería altamente a-helicoidal andamio de proteínas derivadas de la apolipoproteína A1. Las caras hidrófobas de la proteína andamio interactúan con los de acilo graso cadenas laterales de la bicapa de lípido, mientras que las regiones polares frente al entorno acuoso. Los análisis de proteínas de membrana en nanodiscos tienen ventajas significativas sobre liposoma porque las partículas son pequeñas, homogéneas y solubles en agua. Además, los métodos bioquímicos y biofísicos normalmente reservados para las proteínas solubles se pueden aplicar, y desde ambos lados de la membrana. En este protocolo visual, se presenta una reconstitución paso a paso de un carácter bienracterizado ABC transportador bacteriano, el macho-2 MalFGK complejo. La formación del disco es un proceso de auto-ensamblaje que depende de las interacciones hidrofóbicas que tienen lugar durante la eliminación progresiva del detergente. Se describen los pasos esenciales y se destaca la importancia de elegir una correcta proteína-lípido con el fin de limitar la formación de agregados y partículas más grandes de liposomas como polidispersos. Calidad simples controles, tal como cromatografía de filtración en gel, electroforesis en gel nativo y la espectroscopia de dispersión de luz dinámica asegurar que los discos han sido correctamente reconstituido.

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A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscos son pequeñas partículas discoidales que incorporan proteínas de membrana en un pequeño parche de doble capa de fosfolípidos. Proporcionamos un protocolo visual que muestra la incorporación paso a paso del transportador de MalFGK2 en un disco.

Un método paso a paso para la reconstitución de un transportador ABC en partículas lipídicas Nanodisc

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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Research

JoVE Journal

Biology

25.3K vistas.  University of British Columbia. Nanodiscos son pequeñas partículas discoidales que incorporan proteínas de membrana en un pequeño parche de doble capa de fosfolípidos. Proporcionamos un protocolo visual que muestra la incorporación paso a paso del transportador de MalFGK2 en un disco.

Video: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expresión, la solubilización de detergente, y purificación de un transportador de membrana, la proteína de resistencia a múltiples fármacos MexB

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Investigación

Biología

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

En la reconstitución in vitro de los activos T. castaneum telomerasa

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Un método simple para imágenes Arabidopsis Uso de hojas perfluorodecalina como un medio de imágenes infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

La reconstitución de un canal Kv en las membranas lipídicas de Estudios estructurales y funcionales

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstitución de una proteína transmembrana, el canal iónico dependiente de voltaje, KvAP, en Gigante vesículas unilamelares para Microscopía y Patch Clamp Estudios

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procedimiento para evaluar la eficacia de floculantes para la eliminación de partículas dispersas en extractos de plantas

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

Un método de transposición eficiente In Vitro por un sistema de belleza durmiente transcripcionalmente regulada en un Vector lentivirales defectuosa integración

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

Un modelo epitelial bronquial de interfaz aire-líquido para la exposición realista y repetida por inhalación a partículas en el aire para pruebas de toxicidad

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...